Method for attachment of biomolecules to medical device surfaces

ABSTRACT

A method for making a medical device having at least one biomolecule immobilized on a substrate surface is provided. One method of the present invention includes immobilizing a biomolecule comprising an unsubstituted amide moiety on a biomaterial surface. Another method of the present invention includes immobilizing a biomolecule on a biomaterial surface comprising an unsubstituted amide moiety. Still another method of the present invention may be employed to crosslink biomolecules comprising unsubstituted amide moieties immobilized on medical device surfaces. Additionally, one method of the present invention may be employed to crosslink biomolecules comprising unsubstituted amide moieties in solution, thereby forming a crosslinked biomaterial or a crosslinked medical device coating.

Cross-Reference to Related Applications

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/001,994 for “Oxidative Method for Attachment ofBiomolecules to Medical Device Surfaces” to Keogh filed Dec. 31,1997,which is a continuation-in-part of U.S. patent application Ser. No.08/635,187 for “Oxidative Method of Attachment of Biomolecules toSurfaces of Medical Devices” to Keogh filed Apr. 25, 1996.

[0002] This application is further a continuation-in-part of U.S. patentapplication Ser. No. 08/984,922 for “Oxidative Method for Attachment ofGlycoproteins or Glycopeptides to Surfaces of Medical Devices” to Keoghfiled Dec. 4, 1997, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/694,535 for “Oxidative Method of Attachment ofGlycoproteins to Surfaces of Medical Devices” to Keogh filed Aug.9,1996, now U.S. Pat. No. 5,728,420 issued Mar. 17, 1998.

[0003] This application is also a continuation-in-part of U.S. patentapplication Ser. No. 09/012,056 for “A Method for Covalent Attachment ofBiomolecules to Surfaces of Medical Devices” to Keogh filed Jan. 22,1998 which is a continuation-in-part of U.S. patent application Ser. No.09/001,994 for “Oxidative Method for Attachment of Biomolecules toMedical Device Surfaces” to Keogh filed Dec. 31, 1997, and is also acontinuation-in-part of U.S. patent application Ser. No. 08/984,922 for“Oxidative Method for Attachment of Glycoproteins or Glycopeptides toSurfaces of Medical Devices” to Keogh filed Dec. 4, 1997.

[0004] This application is additionally a continuation-in-part of U.S.patent application Ser. No. 09/010,906 for “A Method for IonicAttachment of Biomolecules to Surfaces of Medical Devices” to Keoghfiled Jan. 22, 1998, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/001,994 for “Oxidative Method for Attachment ofBiomolecules to Medical Device Surfaces” to Keogh filed Dec. 31, 1997,and is also a continuation-in-part of U.S. patent application Ser. No.08/984,922 for “Oxidative Method for Attachment of Glycoproteins orGlycopeptides to Surfaces of Medical Devices” to Keogh filed Dec. 4,1997. All the foregoing patent applications and patent are herebyincorporated by reference herein, each in its respective entirety.

BACKGROUND OF THE INVENTION

[0005] For many years, a number of medical devices (e.g., pacemakers,vascular grafts, stents, heart valves, etc.) that contact bodily tissueor fluids of living persons or animals have been developed,manufactured, and used clinically. A major problem with such articles isthat their surfaces tend to adsorb a layer of proteins from tissues andfluids such as tears, urine, lymph fluid, blood, blood products, andother fluids and solids derived from blood. The composition andorganization of this adsorbed protein layer is thought to influence, ifnot control, further biological reactions. Adverse biological reactionssuch as thrombosis and inflammation can diminish the useful lifetime ofmany devices.

[0006] Implantable medical devices also tend to serve as foci forinfection of the body by a number of bacterial species. Thesedevice-associated infections are promoted by the tendency of theseorganisms to adhere to and colonize the surface of the device.Consequently, it has been of great interest to physicians and themedical industry to develop surfaces that are less prone in promotingthe adverse biological reactions that typically accompany theimplantation of a medical device.

[0007] One approach for minimizing undesirable biological reactionsassociated with medical devices is to attach various biomolecules totheir surfaces for the attachment and growth of a cell layer which thebody will accept. Biomolecules such as growth factors, cell attachmentproteins, and cell attachment peptides have been used for this purpose.In addition, biomolecules such as antithrombogenics, antiplatelets,anti-inflammatories, antimicrobials, and the like have also been used tominimize adverse biomaterial-associated reactions.

[0008] A number of approaches have been suggested to attach suchbiomolecules. These approaches typically require the use of couplingagents such as glutaraldehyde, cyanogen bromide, p-benzoquinone,succinic anhydrides, carbodiimides, diisocyanates, ethyl chloroformate,dipyridyl disulphide, epichlorohydrin, azides, among others, which serveas attachment vehicles for coupling of biomolecules to substratesurfaces. For example, covalent attachment of biomolecules using watersoluble carbodiimides is described by Hoffman et al., “Covalent Bindingof Biomolecules to RadiationGrafted Hydrogels on Inert PolymerSurfaces,” Trans. Am. Soc. Artif. Intern. Organs, 18, 10-18 (1972); andIto et al., “Materials for Enhancing Cell Adhesion by Immobilization ofCell-Adhesive Peptide,” J. Biomed. Mat. Res., 25,1325-1337 (1991).

[0009] One type of biomolecule which is commonly coupled to biomaterialsurfaces with coupling molecules is protein. Proteins are polypeptidesmade up of amino acid residues. A protein comprising two or morepolypeptide chains is called an oligomeric protein. In general,established coupling procedures couple proteins to substrate surfacesthrough a protein's lysine amino acid residues which comprise terminalamine moieties. However, not all biomolecules, including some proteinsand peptides, comprise terminal amine moieties. In addition, a number ofestablished coupling procedures couple biomolecules which comprisereactive moieties capable of forming bonds with amine moieties tosubstrate surfaces which comprise terminal amine moieties.

[0010] Thus, what is needed are methods for creating terminal aminemoieties within biomolecules which lack terminal amine moieties. Thesenewly formed terminal amine moieties can then be used to attach thesemodified biomolecules to a medical device substrate surface whichcomprises chemical moieties capable of forming bonds with aminemoieties. In addition, methods are needed for creating terminal aminemoieties on medical device substrate surfaces which lack terminal aminemoieties. These newly formed terminal amine moieties can then be used toattach biomdlecules which comprise chemical moieties capable of formingbonds with amine moieties.

[0011] In some cases, covalently coupling of a biomolecule to abiomaterial surface is not desirable. Therefore, there also exists aneed for methods which may ionically couple a biomolecule to abiomaterial surface. In fact, ionic coupling techniques have anadvantage of not altering the chemical composition of an attachedbiomolecule, thereby reducing the possibility of destroying thebiological properties of an attached biomolecule. Ionic coupling ofbiomolecules also has an advantage of releasing the biomolecule underappropriate conditions. One example of the ionic attachment of abiomolecule to a surface is set forth in U.S. Pat. No. 4,442,133 toGreco et al. In this case, a tridodecyl methylammonium chloride (TDMAC)coating is used to ionically bind an antibiotic agent.

[0012] Another type of biomolecule which is often coupled to biomaterialsurfaces is heparin. Heparin, an anionic biomolecule, is of greatinterest to a number of investigators for the development ofnon-thrombogenic bloodcontact biomaterial surfaces. Heparin, anegatively charged glycosaminoglycan, inhibits blood coagulationprimarily by promoting the activity of antithrombin III (ATIII) to blockthe coagulation enzymes thrombin, factor Xa and, to some extent, factorsIXa, XIa and XIIa. Surfaces bearing bound heparin have been shown tohave anticoagulant activity, therefore, heparinization tends to be apopular technique for improving the thromboresistance of biomaterials.In fact, surface heparinization through an ionic bond is one of themethods used to improve the blood compatibility of a variety ofbiomaterial surfaces.

[0013] The original method of heparinization of surfaces was describedby Gott et al., “Heparin Binding On Colloidal Graphite Surfaces”,Science, 142, 1297-1298 (1963). They prepared agraphite-benzalkonium-heparin surface and observed good non-thrombogeniccharacteristics. Others followed, treating materials with quaternaryammonium salts to ionically bind heparin. Improving on Gott's technique,Grode et al., “Nonthrombogenic Materials via a Simple Coating Process”,Trans. Amer. Soc. Artif. Intern. Organs, 15, 1-6 (1969), eliminated theneed for a graphite coating by using tridodecyl methylammonium chloride(TDMAC). Various other quaternary ammonium salts have also been usedsuch as benzalkonium chloride, cetylpyrrdinium chloride,benzyldimethylstearyammonium chloride, benzylcetyidimethylammoniumchloride as set forth in U.S. Pat. No. 5,069,899 to Whitbourne andMangan.

[0014] Glutaraldehyde was even used by some investigators to increasethe stability of heparin bound ionically through various ammoniumgroups. Rather than using a low molecular weight quaternary salt orquaternary amine, many investigators incorporated the quaternizableamine directly onto substrates by copolymerization techniques. Inanother approach, Barbucci et al., “Surface-Grafted HeparinizableMaterials”, Polymer. 26,1349-1352 (1985), grafted tertiary aminopolymers of poly(amido-amine) structure onto substrates for ionicallycoupling heparin. The cationic amino groups are capable of interactingelectrostatically with the negatively charged groups present in theheparin molecule. They found that the surface's capacity to retainheparin was directly related to the basicity of the grafted cationicamino groups. The greater the basicity of the surface amino groups onthe surface, the greater the capacity of the surface has to retainheparin due to a greater percentage of the surface amino groups beingprotonated at physiological pH.

[0015] Current techniques for immobilization of heparin or other chargedbiomolecules by an ionic bond have been achieved by introducing oppositecharges on a biomaterial surface. The main limit to utilization ofionically bonding methods is the creation of opposing charges on eithera biomolecule or a biomaterial surface or both. Thus, what is needed aremethods for creating charges on a biomolecule or a biomaterial surfaceor both. These newly formed charges can then be used to attach abiomolecule to a medical device substrate surface.

SUMMARY OF THE INVENTION

[0016] The present invention provides methods for attaching abiomolecule to a substrate surface and corresponding medical devices.The present invention provides methods for making a medical devicehaving at least one biomolecule immobilized on a biomaterial surface.One method of the present invention includes converting a biomoleculecomprising an unsubstituted amide moiety (RCONH₂) into anamine-functional material (RNH₂); combining the amine-functionalmaterial with a medical device biomaterial surface comprising a chemicalmoiety (such as, for example, an aldehyde moiety, an epoxide moiety, anisocyanate moiety, a phosphate moiety, a sulphate moiety or acarboxylate moiety) which is capable of forming a chemical bond with theamine-functional material, to bond the two materials together to form animmobilized biomolecule on a medical device biomaterial surface.

[0017] The present invention provides another method for making amedical device having at least one biomolecule immobilized on abiomaterial surface. The method includes converting a medical devicebiomaterial surface comprising an unsubstituted amide moiety (RCONH₂)into an amine-functional material (RNH₂); combining the amine-functionalmaterial with a biomolecule comprising a chemical moiety (such as, forexample, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, aphosphate moiety, a sulphate moiety or a carboxylate moiety) which iscapable of forming a chemical bond with the amine-functional material,to bond the two materials together to form an immobilized biomolecule ona medical device biomaterial surface.

[0018] The present invention also provides a method for making a medicaldevice having at least one biomolecule immobilized on a biomaterialsurface. The method includes converting a biomolecule comprising anunsubstituted amide moiety (RCONH₂) into an amine-functional material(RNH₂); converting the amine-functional material into aguanidino-functional material (RNHC(NH)NH₂); combining theguanidino-functional material with a medical device biomaterial surfacecomprising a chemical moiety (such as, for example, a 1,2-dicarbonylmoiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety)which is capable of forming a chemical bond with theguanidino-functional material, to bond the two materials together toform an immobilized biomolecule on a medical device biomaterial surface.

[0019] Additionally, the present invention provides a method for makinga medical device having at least one biomolecule immobilized on abiomaterial surface. The method includes converting a medical devicebiomaterial surface comprising an unsubstituted amide moiety (RCONH₂)into an aminefunctional material (RNH₂); converting the amine-functionalmaterial into a guanidino-functional material (RNHC(NH)NH₂); combiningthe guanidinofunctional material with a biomolecule comprising achemical moiety (such as, for example, a 1,2-dicarbonyl moiety, aphosphate moiety, a sulphate moiety or a carboxylate moiety) which iscapable of forming a chemical bond with the guanidino-functionalmaterial, to bond the two materials together to form an immobilizedbiomolecule on a medical device biomaterial surface.

[0020] Another method of the present invention may be employed tocrosslink biomolecules, located in solution or on biomaterial surfaces,comprising both an unsubstituted amide moiety and a chemical moietycapable of forming a chemical bond with an amine moiety. Such a methodcomprises converting a biomolecule comprising an unsubstituted amidemoiety into an amine-functional material; allowing the amine-functionalmaterial to combine with the chemical moiety capable of forming achemical bond with an amine moiety to form a chemical linkage and acrosslinked material. This crosslinked material may be employed as abiomaterial or as a biomaterial coating. In addition, such crosslinkedmaterials may be further modified to contain additional biomolecules.For example, biomolecules comprising a chemical moiety capable offorming a chemical bond with an amine moiety may be attached to residualamine moieties present in or on the surface of the crosslinked material.Alternatively, biomolecules comprising an amine moiety may be attachedto residual chemical moieties capable of forming chemical bonds withamine moieties present in or on the surface of the crosslinked material.Additionally, biomolecules coated onto a biomaterial surface may becrosslinked according to still another method of the present invention.

[0021] Another method of the present invention may be employed tocrosslink biomolecules, located in solution or on biomaterial surfaces,comprising both an unsubstituted amide moiety and a chemical moietycapable of forming a chemical bond with a guanidino moiety. Such amethod comprises converting a biomolecule comprising an unsubstitutedamide moiety into an amine-functional material; converting theamine-functional material into a guanidino-functional material(RNHC(NH)NH₂); allowing the guanidino-functional material to combinewith the chemical moiety capable of forming a chemical bond with anguanidino moiety to form a chemical linkage and a crosslinked material.This crosslinked material may be employed as a biomaterial or as abiomaterial coating. In addition, such crosslinked materials may befurther modified to contain additional biomolecules. For example,biomolecules comprising a chemical moiety capable of forming a chemicalbond with an guanidino moiety may be attached to residual guanidinomoieties present in or on the surface of the crosslinked material.Alternatively, biomolecules comprising a guanidino moiety may beattached to residual chemical moieties capable of forming chemical bondswith guanidino moieties present in or on the surface of the crosslinkedmaterial. Additionally, biomolecules coated onto a biomaterial surfacemay be crosslinked according to still another method of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0022] As used in the specification and claims hereof, the followingterms have the particular meanings and definitions set forth below.

[0023] We define the term “chemical bond” appearing herein to beinterpreted broadly to encompass not only covalent bonding and ionicbonding but also interactions, such as, for example, van der Waalsforces and hydrogen bonding.

[0024] We define the term “biomolecule” appearing herein as a materialthat engages in a biological activity or which is effective inmodulating a biological activity such as eliminating, reducing orenhancing various biological reactions that typically accompany theexposure of human or animal bodily tissues or fluids to a biomaterial.Biomaterial-associated reactions include thrombosis, tissue death, tumorformation, allergic reaction, foreign-body reaction (rejection),inflammatory reaction, infection and cellular attachment and growth.Biomolecules suitable for use in the present invention comprise eitheran unsubstituted amide moiety, a 1,2-dihydroxy moiety, a 2-aminoalcoholmoiety, a 1,2-dicarbonyl moiety, a guanidino moiety, a chemical moietycapable of forming either a covalent bond with an amine moiety (such as,for example, an aldehyde moiety, an epoxide moiety or an isocyanatemoiety) or a chemical moiety capable of forming an ionic bond with anamine moiety (such as, for example, a phosphate moiety, a sulphatemoiety or a carboxylate moiety), or any possible combination of any oneor more of these moieties alone or in combination. In addition, the term“biomolecule” appearing herein may mean any one or more of a biomoleculealone or a combination of different biomolecules.

[0025] Generally, biomolecules used according to this invention may be,for example an anticoagulant agent such as heparin and heparan sulfate,an antithrombotic agent, a clotting agent, a platelet agent, ananti-inflammatory agent, an antibody, an antigen, an immunoglobulin, adefense agent, an enzyme, a hormone, a growth factor, aneurotransmitter, a cytokine, a blood agent, a regulatory agent, atransport agent, a fibrous agent, a protein such as avidin, aglycoprotein, a globular protein, a structural protein, a membraneprotein and a cell attachment protein, a peptide such as a glycopeptide,a structural peptide, a membrane peptide and a cell attachment peptide,a proteoglycan, a toxin, an antibiotic agent, an antibacterial agent, anantimicrobial agent such as penicillin, ticarcillin, carbenicillin,ampicillin, oxacillian, cefazolin, bacitracin, cephalosporin,cephalothin, cefuroxime, cefoxitin, norfloxacin, perfloxacin andsulfadiazine, hyaluronic acid, a polysaccharide, a carbohydrate, a fattyacid, a catalyst, a drug, biotin, a vitamin, a DNA segment, a RNAsegment, a nucleic acid, a lectin, a ligand and a dye (which acts as abiological ligand). The biomolecules may be found in nature (naturallyoccurring) or may be chemically synthesized.

[0026] Biomolecules may be chemically synthesized by a number of methodswell known to those skilled in the art. For example, a number of methodsare know for synthesizing proteins or peptides from amino acidsincluding solution (classical) synthesis methods and solid phase (e.g.,SPPS) synthesis methods. Peptides of varying length may also be formedby the partial hydrolysis of very long polypeptide chains of proteins.In addition, proteolytic enzymes such as trypsin, chymotrypsin, andpepsin may be used to cleave specific peptide bonds in proteins andpeptides. Furthermore, site-specific oxidative cleavage of peptide bondsusing either cupric ions or ferric ions may be used to create peptideand/or polypeptide chains comprising unsubstituted amide moieties.Peptides are short chains constructed of two or more amino acidscovalently joined through substituted amide linkages, termed peptidebonds. Two amino acids joined by a peptide bond forms a dipeptide. Threeamino acids joined by two peptide bonds forms a tripeptide; similarly,there are tripeptides and pentapeptides. When there are many amino acidsjoined together, the structure is termed a polypeptide. In general,polypeptides contain less than 100 amino acid residues and proteinscontain 100 or more amino acid residues.

[0027] Some biomolecules are susceptible to conformational changes whenbrought into contact with a hydrophobic substrate surface. Theseconformational changes can lead to the exposure of internalized nonpolargroups which may lead to hydrophobic interactions between thebiomolecule and the surface. These hydrophobic interactions may causethe exclusion of water molecules that normally surround the biomoleculein solution. This exclusion of water molecules between the biomoleculeand the surface strengthens the hydrophobic interaction and may causefurther conformational change of the biomolecule. The degree ofconformational change a biomolecule experiences may or may not destroyits biological properties. Therefore, one must take into account thehydrophobic nature of the substrate surface when attaching biomoleculeswhich are prone to hydrophobic interactions. In such cases, it ispreferred to create a hydrophilic environment on the biomaterialsurface, thereby preventing any unwanted hydrophobic interactionsbetween the biomolecule and the surface which may destroy the biologicalproperties of the biomolecule.

[0028] There are a number of surface-derivatization techniques (e.g.,grafting techniques) well known to those skilled in the art for creatinghydrophilic substrate surfaces. For example, techniques based on cericion initiation, ozone exposure, corona discharge, UV irradiation andionizing radiation (⁶⁰Co, X-rays, high energy electrons, plasma gasdischarge) are known.

[0029] We define the term “glycoprotein” appearing herein as aconjugated protein which contains at least one carbohydrate group whichmay comprise a 1,2-dihydroxy moiety. A typical glycoprotein contains oneor more oligosaccharide units linked to either asparagine amino acidresidues by N-glycosidic bonds or serine or threonine amino acidresidues by O-glycosidic bonds. The saccharide unit directly bonded toasparagine is typically Nacetylglucosamine, whereasN-acetylgalactosamine tends to be the saccharide unit bonded to serineor threonine residues. Oligosaccharides bound to glycoproteins maycontain a variety of carbohydrate units. They tend to be located atsites away from the biologically active site of the protein.

[0030] Thus, oligosaccharide moieties of glycoproteins can typically bemodified with little or no effect on the biological properties of theprotein We define the term “glycopeptide” appearing herein as aconjugated peptide which contains at least one carbohydrate group whichmay comprise a 1,2-dihydroxy moiety. As mentioned earlier, peptides areshort chains constructed of two or more amino acids covalently joinedthrough substituted amide linkages, termed peptide bonds. Two aminoacids joined by a peptide bond forms a dipeptide. Three amino acidsjoined by two peptide bonds forms a tripeptide; similarly, there aretetrapeptides and pentapeptides. When there are many amino acids joinedtogether, the structure is termed a polypeptide. In general,polypeptides contain less than 100 amino acid residues and proteinscontain 100 or more amino acid residues.

[0031] Glycoproteins and glycopeptides can be chemically synthesized bya number of methods well known to those skilled in the art. For example,glycoproteins and/or glycopeptides can be formed from natural orchemically synthesized proteins and/or peptides by glycosylation, whichis the addition of carbohydrate side chains. There are a number ofmethods well known to those skilled in the art for glycosylatingproteins or peptides. For example, side-chain glycosylation can beperformed chemically with glycosylbromides for serine (Ser, S) andthreonine (Thr, T) amino acid residues and glycosylamines for asparticacid (Asp, D) amino acid residues, thereby producing glycosylatedasparagine (Asn, N) amino acid residues. In addition, glycosylatingenzymes can be used to attach carbohydrate side chains to proteins orpeptides.

[0032] Proteins or peptides, chemically synthesized or naturallyoccurring, also suitable for use in the present invention comprise anasparagine (Asn, N) amino acid residue or a glutamine (Gln, Q) aminoacid residue, both of which comprise an unsubstituted amide moiety. Inaddition, proteins or peptides, again chemically synthesized ornaturally occurring, which are also suitable for use in the presentinvention comprise a N-terminal serine (Ser, S) amino acid residue, aN-terminal threonine (Thr, T) amino acid residue, or a 5-hydroxylysine(5-hydroxylysine is only known to occur naturally in collagen, but inprincipal may be placed anywhere in a synthetic peptide or protein)amino acid residue, all of which comprise a 2-aminoalcohol moiety.

[0033] Biomolecules or biomaterials of the present invention comprisingan unsubstituted amide moiety may be converted into an amine-functionalmaterial via a Hofmann rearrangement reaction, also known as a Hofmanndegradation of amides reaction. For example, Wirsen et al., “Bioactiveheparin surfaces from derivatization of polyacrylamide-grafted LLDPE”,Biomaterials, 17, 1881-1889 (1996), demonstrated the conversion ofunsubstituted amide moieties of a polyacrylamide-low densitypolyethylene film into primary amine moieties using a Hofmannrearrangement reaction. In another example, Sano et al., “Introductionof functional groups onto the surface of polyethylene for proteinimmobilization”, Biomaterials. 14, 817-822 (1993), demonstrated theconversion of unsubstituted amide moieties of a polyacrylamide-highdensity polyethylene film into primary amine moieties using a Hofmannrearrangement reaction. In another example, Fuller et al., “A new classof amino acid based sweeteners”, J. Am. Chem. Soc., 107, 5821-5822(1985), demonstrated the conversion of an unsubstituted amide moiety ofan amino acid into a primary amine moiety using a Hofmann rearrangementreaction. In another example, Loudon et al., “Conversion of aliphaticamides into amines with [I,I-bis(trifluoroacetoxy)iodo]benzene. 1. Scopeof the reaction”, J. Org. Chem., 49, 4272-4276 (1984), demonstrated theconversion of various unsubstituted amide moieties, including the amideside chain in a glutamine amino acid residue, into primary aminemoieties using Hofmann rearrangement reactions.

[0034] The Hofmann rearrangement reaction which converts anunsubstituted amide moiety into a primary amine moiety may be carriedout with chemical reactants such as, for example, bromine, bromide,bromite, hypobromite, chlorine, chloride, chlorite, hypochlorite, leadtetraacetate, benzyltrimethylammonium tribromide and hypervalentorganoiodine compounds such as, for example,[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene andiodosylbenzene. A general discussion of Hofmann rearrangement reactionsis contained in Comprehensive Organic Synthesis, Volume 6, 800-806,Pergamon Press. For example, Kajigaeshi et al., “An efficient method forthe Hofmann degradation of amides by use of benzyltrimethylammoniumtribromide”, Chemistry Letters, 463-464 (1989), described variousmethods for obtaining amines from amides using the Hofmann rearrangementreaction. These methods include, for example, the use of bromine orchlorine in an alkaline solution, the use of lead tetraacetate in analcohol solution, the use of [bis(trifluoroacetoxy)iodo]benzene in anaqueous acetonitrile solution, the use of sodium bromite in an alkalinesolution, and the use of benzyltrimethylammonium tribromide in analkaline solution. Catalysts such as, for example, triethylamine,tin(IV) chloride, dibutylstannyl dilaurate or pyridine are sometimesused in a Hofmann rearrangement reaction. Typical solvents include, forexample, water, hydroxides, methoxides, alcohols, dimethylformamide,acetonitrile, benzene, carboxylic acids or combinations thereof.

[0035] Depending on the chemical reactants, the Hofmann rearrangementreaction may be carried out under acidic, neutral or basic conditions.Although applicants do not wish to be bound by any single theory, it isgenerally believed that when the reaction is carried out with particularamine forming agents in an aqueous base a N-halo amide intermediate isformed. An isocyanate is then formed from the N-halo amide intermediate.The formed isocyanate then readily hydrolyzes into a primary amine. Incontrast, when the reaction is carried out in an alcohol a carbamate isgenerally formed. The carbamate may then be hydrolyzed into a primaryamine. For example, when the reaction of an amide with bromine iscarried out in methanol containing sodium methoxide instead of inaqueous base, the product is a carbamate which is easily converted to anamine via hydrolysis. Depending on the reaction conditions,side-reactions such as chain scission, hydrolysis and/or urea formationmay occur. However, side-reactions may be minimized by changes in thereaction conditions. For example, changes in the reaction conditionssuch as pH, time, temperature and/or the amount of amine forming agentmay minimize various side-reactions.

[0036] In general, the Hofmann rearrangement reaction is carried outwith an amine forming agent in amounts ranging from about 0.5 eq. toabout 2 eq. based on the amide content of the biomolecule orbiomaterial. In addition, the reaction is generally carried out at atemperature between about −10 and about 100 degrees Celsius, preferablyfrom about 0 and about 50 degrees Celsius. Depending on the material, aHofmann rearrangement reaction may be carried out for as short as a fewminutes to as long as many hours. Time, temperature and pH limitationsof the present invention are generally governed by the stability of thematerials imparted by the Hofmann rearrangement process. Wide latitudemay be employed in determining the optimum conditions for a particularsystem. Such conditions may be determined readily by one skilled in theart by routine experimentation upon examination of the informationpresented herein.

[0037] We define the term “amine forming agent” appearing herein toinclude any chemical agent or combination of chemical agents capable offorming an amine moiety upon its or their reaction with an unsubstitutedamide moiety. Examples of amine forming agents include, for example,bromine, bromide, bromite, hypobromite, chlorine, chloride, chlorite,hypochlorite, lead tetraacetate, benzyltrimethylammonium tribromide andhypervalent organoiodine compounds such as, for example,[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene andiodosylbenzene. Amine forming agents include any of the many possibleHofmann rearrangement reactants. As mentioned above, the term “amineforming agent” appearing herein may mean any one or more of an amineforming agent or a combination of different amine forming agents.

[0038] Biomolecules or biomaterials of the present invention comprising,in addition to an unsubstituted amide moiety, a primary amine moiety ofwhich is desired to be left intact and unreacted following coupling maybe protected or blocked prior to the Hofmann rearrangement reaction. Forexample, a protein may comprise both a lysine amino acid residue and,for example, an asparagine amino acid residue. As mentioned earlier,there are a number of established coupling procedures which may couple aprotein comprising a lysine amino acid residue to a substrate surfacethrough the protein's lysine amino acid residue. However, a protein'slysine amino acid residues are typically associated with the protein'sbiologically active site. Therefore, coupling a protein to a substratesurface via a protein's primary amine moiety in the side chain of itslysine amino acid residue may destroy the biological properties of theattached protein. However, a protein's lysine amino acid residue may beprotected or blocked by a number of methods well known to those skilledin the art. For example, the amine moiety may be protected using, forexample, a tert.butyloxycarbonyl (Boc) group which is typically cleavedwith acid, a benzyloxycarbonyl (Z) group which is typically cleaved byhydrogenolysis, a biphenylisopropyloxycarbonyl (Bpoc) group which istypically cleaved with acid, a triphenylmethyl (trityl) group which istypically cleaved with acid, a 9-fluoroenylmethyloxycarbonyl (Fmoc)group which is typically cleaved with base or a blocking group which ispH stable but is cleaved by enzyme-catalyzed hydrolysis. The appropriateamine-blocking group to use to protect the amine moiety will dependhighly on the entire sequence of reaction conditions chosen forbiomolecule attachment or crosslinking. Following blocking of the aminemoiety of the lysine residue, the amide moiety of the asparagine residuemay then be converted into an amine moiety via a Hofmann rearrangementreaction. The protein may then be coupled to the substrate via onemethod of the present invention through the newly formed amine moiety.Following coupling, the amine-blocking group may then be removed,thereby preserving the protein's biological activity. This type ofblocking scheme may be employed on biomolecules and/or biomaterials ofthe present invention which contain amine moieties which are desired tobe left intact and unreacted.

[0039] Biomaterials of the present invention not comprisingunsubstituted amides on their surface may be amidated readily through anumber of methods well known in the art. For example, unsubstitutedamides may be provided by ceric ion grafting acrylamide to a biomaterialsurface as set forth in U.S. Pat. No. 5,344,455 to Keogh et al.Alternatively, for example, a grafted acrylamide-containing polymer maybe attached by radiation grafting as set forth in U.S. Pat. No.3,826,678 to Hoffman et al. There are a number of surface-derivatizationtechniques (e.g., grafting techniques) well known in the art forcreating substrate surfaces comprising unsubstituted amide moieties. Forexample, techniques based on ceric ion initiation, ozone exposure,corona discharge, UV irradiation and ionizing radiation (⁶Co, X-rays,high energy electrons, plasma gas discharge) are known. In addition,amides can generally be prepared by reaction of ammonia with acidchlorides. This reaction is commonly known as ammonolysis. Acidchlorides are prepared by substitution Cl for the —OH group of acarboxylic acid. Reagents commonly used to form acid chlorides fromcarboxylic acids include thionyl chloride (SOCl₂), phosphorustrichloride (PCl₃) and phosphorus pentachloride (PCl₅). Two amino acidscomprising carboxylic acid moieties which may be converted into acidchlorides are aspartic acid (Asp, D) amino acid and glutamic acid (Glu,E) amino acid. The acid chloride moieties may then be converted intoamide moieties followed by conversion into amine moieties. In addition,treatment of an ester moiety with ammonia, generally in ethyl alcoholsolution, will yield an amide moiety.

[0040] Biomolecules or biomaterials suitable for use according to onemethod of the present invention may comprise at least one negativelycharged moiety (also known as an anionic moiety) at physiological pH,such as a phosphate moiety, a sulphate moiety or a carboxylate moiety. Anegatively charged moiety is capable of interacting electrostaticallywith a positively charged moiety (also known as a cationic moiety)thereby forming an ionic chemical bond or linkage.

[0041] Biomaterials that do not contain a negative charge on theirsurfaces may be furnished with a net negative and may be modifiedreadily through a number of methods well known in the art. For example,polyethylene may be exposed to sulfuric acid comprising potassiumpermanganate thereby creating a negative charge. Other examples offurnishing biomaterials with negatively charged surfaces are taught inU.S. Pat. No. 5,344,455 to Keogh et al. and U.S. Pat. No. 5,429,618 toKeogh.

[0042] Biomolecules or biomaterials suitable for use according to onemethod of the present invention may comprise at least one positivelycharged moiety at physiological pH, such as an amine moiety or aguanidino moiety. Biomolecules or biomaterials that do not comprise apositive charge may be furnished with a net positive charge by onemethod of the present invention. As mentioned earlier, a positivelycharged moiety is capable of interacting electrostatically with anegatively charged moiety thereby forming an ionic chemical bond orlinkage.

[0043] Biomolecules or biomaterials suitable for use according to onemethod of the present invention may comprise at least one epoxidemoiety. Epoxide moieties are three-membered rings comprising two carbonatoms and an oxygen atom. An epoxide ring is also known as an oxiranering. There are a number of techniques well known in the art to produceepoxides. Epoxide moieties are highly reactive due to the ease ofopening of the highly strained three-membered ring. An epoxide moietywill react readily with an amine moiety, thereby forming a covalent bondor linkage. The reaction of an epoxide moiety with an amine moiety iswell known in the art.

[0044] Biomolecules or biomaterials suitable for use according to onemethod of the present invention may comprise at least one isocyanatemoiety. An isocyanate moiety (RNCO) will react readily with an aminemoiety, thereby forming a covalent bond or linkage called a urea. Thereare a number of techniques well known in the art to produce isocyanates.In addition, the reaction of an isocyanate moiety with an amine moietyis well known in the art.

[0045] We define the term “1,2-dihydroxy moiety” appearing herein as acarbon-carbon bond bearing two adjacent hydroxyl moieties.

[0046] We define the term “2-aminoalcohol moiety” appearing herein as acarbon-carbon bond bearing an amine moiety adjacent to a hydroxylmoiety.

[0047] The 1,2-dihydroxy moiety and the 2-aminoalcohol moiety are bothoxidizable with periodate, which may be provided as periodic acid orsalts thereof, such as sodium periodate, potassium periodate, or otheralkali metal periodates. Typically, a stoichiometric amount of periodateis used to oxidize the desired number of 1,2-dihydroxy moieties or2-aminoalcohol moieties to form aldehyde moieties, however less than astoichiometric amount or more than a stoichiometric amount may be used.Periodate oxidation of a 1,2-dihydroxy moiety or a 2-aminoalcohol moietyis generally carried out in an aqueous solution, preferably an aqueousbuffered solution, at a temperature that does not destroy the desiredproperties of the material. Generally, buffers having a pH in a rangebetween about 4 and about 9 can be used, with a pH between about 6 andabout 8 desired for certain pH sensitive materials. Generally, theoxidation is carried out at a temperature between about 0 and about 50degrees Celsius, and preferably at a temperature between about 4 andabout 37 degrees Celsius.

[0048] Depending on the material, oxidation reactions can be carried outfor as short as a few minutes to as long as many days. Commonly,oxidation is complete within 24 hours. Long-term oxidation reactions arepreferably performed in the dark to prevent “overoxidation.”

[0049] Treatment times and temperatures for the oxidation process tendto be inversely related. That is, higher treatment temperatures requirerelatively shorter treatment times. Time and temperature limitations ofthe present invention are generally governed by the stability of thematerials imparted by the oxidation process. Wide latitude may beemployed in determining the optimum conditions for a particular system.Such conditions may be determined readily by one skilled in the art byroutine experimentation upon examination of the information presentedherein.

[0050] Subsequent to oxidation, the reaction solution may be storedprior to use at about 4 degrees Celsius. Typically, the storagestability of the reaction solution at a neutral pH or slightly acidic pHmay extend between about one and about fourteen days and sometimes evenmonths when stored in the dark.

[0051] In general, an aldehyde moiety (RCHO) will react chemically witha primary amine moiety (R′NH₂) to form a relatively unstable iminemoiety (R′N═CHR). The reaction of an aldehyde moiety with a primaryamine moiety which is commonly referred to as a Schiff base reaction maybe carried out under the same conditions as described above for theperiodate oxidation reaction, which is generally designed to protect abiomolecule from damage.

[0052] To stabilize the relatively unstable imine linkage, subsequentreductive alkylation of the imine moiety is carried out using reducingagents (i.e., stabilizing agents) such as, for example, sodiumborohydride, sodium cyanoborohydride, and amine boranes, to form asecondary amine (R′NH—CH₂R). This reaction can also be carried out underthe same conditions as described above for the periodate oxidationreaction. Typically, however, the coupling and stabilizing reactions arecarried out in a neutral or slightly basic solution and at a temperaturebetween about 0 and about 50 degrees Celsius. Preferably, the pH isbetween about 6 and about 10, and the temperature is between about 4 andabout 37 degrees Celsius, for the coupling and stabilizing reactions.These reactions (coupling and stabilizing) may be allowed to proceed forjust a few minutes or for many hours.

[0053] Commonly the reactions are complete (i.e., coupled andstabilized) within 24 hours.

[0054] We define the term “guanidino moiety” appearing herein to includeguanidine, guanidinium, guanidine derivatives such as (RNHC(NH)NHR′),monosubstituted guanidines, monoguanides, biguanides, biguanidederivatives such as (RNHC(NH)NHC(NH)NHR″), and the like. In addition,the term “guanidino moiety” appearing herein may mean any one or more ofa guanide alone or a combination of different guanides.

[0055] Guanidine is the imide of urea, or the amidine of carbamic acid.It is a very strong base with a pKa of 13.5 in water. The great basicityof guanidine is a result of the stability of the conjugated acid(guanidinium) in water. The positive charge on the guanidinium ion canbe spread equally among the three nitrogens by resonance. Theguanidinium ion is also quite hydrophilic and is well solvated inaqueous media due to the extensive hydrogen bonding of six potentialhydrogen bond donors to the solvent. The partial positive charge of thehydrogen bond donors increases their strength for donation to thenegative dipole of water. Crystal structures of simple guanidiniumderivatives have revealed several common features. First, the C—N singlebond length in an alkyl guanidine is typically shorter than the usualC—N single bond length. Usually, the three C—N bonds in the guanidiniumgroup itself are nearly equal in length with an average of 1.33 A. Thethree N—C—N bond angles are almost always near 1200.

[0056] The guanidinium group's features make it a very attractivemoiety. For example, its high basicity (a pK_(a) of 13.5 for guanidiniumitself) allows it to remain protonated over a much wider range of pHthan does the ammonium group. In fact, at physiological pH, all but asmall fraction of the guanidine molecules will exist as positivelycharged species. The guanidinium group's enhanced hydrogen bondingcapabilities, typically two linear hydrogen bonds, allow it to formtighter complexes with anions that are capable of hydrogen bonding. Infact, the guanidinium group may form characteristic pairs ofzwitterionic hydrogen bonds which provide binding strength by theircharge and structural organization by their arrangement. Another featureof guanidines are their ability to react with 1,2-dicarbonyl moietiesunder mild alkaline conditions to form covalent bonds. The reaction of aguanidino moiety and a 1,2-dicarbonyl moiety is similar to a Schiff basereaction (the reaction between an amine moiety and an aldehyde moiety).In some cases, it may be desirable to use a stabilizing agent such asborate ion (BO₃ ⁻) to stabilize the resultant compound.

[0057] We define the term “1,2-dicarbonyl moiety” appearing herein astwo carbonyl (C═O) groups located on adjacent carbon atoms. A carbonylgroup contains a carbon-oxygen double bond.

[0058] Biomolecules or biomaterials of the present invention comprisingan unsubstituted amide moiety may be modified to comprise guanidinomoieties.

[0059] The method of the present invention includes converting anunsubstituted amide moiety (RCONH₂) into an amine-functional material(RNH₂). The amine-functional material is then modified to compriseguanidino moieties by reaction with compounds such as S-ethylthiouroniumbromide, S-ethylthiouronium chloride, O-methylisourea,O-methylisouronium sulfate, O-methylisourea hydrogen sulfate,S-methylisothiourea, 2-methyl-1-nitroisourea, aminoiminomethanesulfonicacid, cyanamide, cyanoguanide, dicyandiamide,3,5-dimethyl-1-guanylpyrazole nitrate and 3,5-dimethyl pyrazole. Forexample, reaction of amines with O-methylisourea, Smethylisourea,S-ethylthiouronium bromide or S-ethylthiouronium chloride, therebyyielding guanidino moieties, are generally completed after 8 hours at 70degrees Celsius in a solution of sodium hydroxide (NaOH) at pH 10.Reactions of amines with aminoiminomethanesulfonic acid or cyanamide aregenerally performed at room temperature. Another example is the reactionof an amine with 2-methyl-1-nitroisourea in water to form anitroguanidine. The nitro group is then easily removed to form aguanidino moiety by hydrogenolysis.

[0060] We define the term “guanidino forming agent” appearing herein toinclude any chemical agent capable of forming a guanidino moiety uponits reaction with a non-guanidino moiety. Examples of guanidino formingagents include S-ethylthiouronium bromide, S-ethylthiouronium chloride,O-methylisourea, O-methylisouronium sulfate, O-methylisourea hydrogensulfate, S-methylisothiourea, 2-methyl-1-nitroisourea,aminoiminomethanesulfonic acid, cyanamide, cyanoguanide, dicyandiamide,3,5-dimethyl-1-guanylpyrazole nitrate and 3,5-dimethyl pyrazole. Inaddition, the term “guanidino forming agent” appearing herein may meanany one or more of a guanidino forming agent or a combination ofdifferent guanidino forming agents.

[0061] We define the term “biomaterial” appearing herein as a materialthat is substantially insoluble in human or animal bodily fluids andthat is designed and constructed to be placed in or onto the body or tocontact fluid of the body. Ideally, a biomaterial will not induceundesirable reactions in the body such as blood clotting, tissue death,tumor formation, allergic reaction, foreign body reaction (rejection) orinflammatory reaction; will have the physical properties such asstrength, elasticity, permeability and flexibility required to functionfor the intended purpose; may be purified, fabricated and sterilizedeasily; will substantially maintain its physical properties and functionduring the time that it remains implanted in or in contact with thebody. Biomaterials suitable for use in the present invention compriseeither an unsubstituted amide moiety, a 1,2-dihydroxy moiety, a2-aminoalcohol moiety, a 1,2-dicarbonyl moiety, a guanidino moiety, achemical moiety capable of forming either a covalent bond with an aminemoiety (such as an aldehyde moiety, an epoxide moiety or an isocyanatemoiety) or a chemical moiety capable of forming an ionic bond with anamine moiety (such as a phosphate moiety, a sulphate moiety or acarboxylate moiety), or any possible combination of any one or more ofthese moieties alone or in combination. Additionally, biomaterialscomprising both an unsubstituted amide moiety and a 1,2-dihydroxymoiety, a 2-aminoalcohol moiety, or a chemical moiety capable of forminga chemical bond with an amine moiety may by crosslinked, according toone method of the present invention. Also, biomaterials may befabricated by crosslinking biomolecules, comprising both anunsubstituted amide moiety and a 1,2-dihydroxy moiety, a 2-aminoalcoholmoiety, or a chemical moiety capable of forming a chemical bond with anamine moiety, according to another method of the present invention.

[0062] Biomaterials or substrates that may be modified according to onemethod of the present invention include metals such as titanium,titanium alloys, TiNi alloys, shape memory alloys, super elastic alloys,aluminum oxide, platinum, platinum alloys, stainless steels, stainlesssteel alloys, MP35N, elgiloy, haynes 25, stellite, pyrolytic carbon,silver carbon, glassy carbon, polymers such as polyamides,polycarbonates, polyethers, polyesters, polyolefins includingpolyethylenes or polypropylenes, polystyrenes, polyurethanes,polyvinylchlorides, polyvinylpyrrolidones, silicone elastomers,fluoropolymers, polyacrylates, polyisoprenes, polytetrafluoroethylenes,rubber, minerals or ceramics such as hydroxapatite, human or animalprotein or tissue such as bone, skin, teeth, collagen, laminin, elastinor fibrin, organic materials such as wood, cellulose, or compressedcarbon, a string, a suture, a fiber, a mesh and other materials such asglass and the like. Biomaterials of the present invention made usingthese materials may be coated or uncoated, porous or nonporous,permeable and nonpermeable, derivatized or underivatized. We define theterm “medical device” appearing herein as a device having surfaces thatcontact human or animal bodily tissue and/or fluids in the course oftheir operation. This definition includes within its scope, for example,extracorporeal devices for use in surgery such as blood oxygenators,blood pumps, blood sensors, tubing used to carry blood and the likewhich contact blood which is then returned to the patient. Thedefinition includes within its scope endoprostheses implanted in bloodcontact in a human or animal body such as vascular grafts, stents,pacemaker leads, heart valves, and the like that are implanted in bloodvessels or in the heart. The definition also includes within its scopedevices for temporary intravascular use such as catheters, guide wires,and the like which are placed into the blood vessels or the heart forpurposes of monitoring or repair.

[0063] One method of the invention may be used to modify substrates ofany shape or form including tubular, sheet, rod and articles of propershape for use in a number of medical devices such as vascular grafts,aortic grafts, arterial, venous, or vascular tubing, vascular stents,dialysis membranes, tubing or connectors, blood oxygenator tubing ormembranes, ultrafiltration membranes, intra-aortic balloons, blood bags,catheters, sutures, soft or hard tissue prostheses, syntheticprostheses, prosthetic heart valves, tissue adhesives, cardiac pacemakerleads, artificial organs, endotracheal tubes, lenses for the eye such ascontact or intraocular lenses, blood handling equipment, apheresisequipment, diagnostic and monitoring catheters and sensors, biosensors,dental devices, drug delivery systems, or bodily implants of any kind.

[0064] The present invention has an object of solving a number ofproblems associated with the use of medical devices. The presentinvention includes within its scope methods for attaching biomoleculesto biomaterial surfaces for use in medical devices. The presentinvention further provides methods for fabricating crosslinkedbiomaterials or crosslinked biomaterial coatings comprisingbiomolecules.

[0065] One preferred method of the present invention may be employed toimmobilize a least one biomolecule comprising at least one2-aminoalcohol moiety on a biomaterial surface comprising at least oneunsubstituted amide moiety. The method comprises the steps of: combininga periodate with a biomolecule comprising a 2-aminoalcohol moiety toform an aldehydefunctional material in an aqueous solution having a pHbetween about 4 and about 9 and a temperature between about 0 and about50 degrees Celsius; providing a biomaterial surface comprising anunsubstituted amide moiety, converting the amide moiety into an aminemoiety using an amine forming agent to form an amine-functionalmaterial; combining the aldehyde-functional material with theamine-functional material to chemically bond the two materials togetherthrough an imine moiety; and reacting the imine moiety with a reducingagent to form an immobilized biomolecule on a medical device biomaterialsurface through a secondary amine linkage.

[0066] Another preferred method of the present invention may be employedto immobilize a least one glycoprotein or glycopeptide comprising atleast one 1,2-dihydroxy moiety on a biomaterial surface comprising atleast one unsubstituted amide moiety. The method includes the steps of:combining a periodate with a glycoprotein or glycopeptide comprising a1,2-dihydroxy moiety to form an aldehyde-functional material in anaqueous solution having a pH between about 4 and about 9 and atemperature between about 0 and about 50 degrees Celsius; providing abiomaterial surface comprising an unsubstituted amide moiety, convertingthe amide moiety into an amine moiety using an amine forming agent toform an amine-functional material; combining the aldehyde-functionalmaterial with the amine-functional material to chemically bond the twomaterials together through an imine moiety; and reacting the iminemoiety with a reducing agent to form an immobilized glycoprotein orglycopeptide on a medical device biomaterial surface through a secondaryamine linkage.

[0067] Still another preferred method of the present invention may beemployed to immobilize a least one biomolecule comprising at least oneepoxide moiety on a biomaterial surface comprising at least oneunsubstituted amide moiety. The method includes the steps of: providinga biomaterial surface comprising an unsubstituted amide moiety,converting the amide moiety into an amine moiety using an amine formingagent to form an amine-functional material; combining theepoxide-functional biomolecule with the amine-functional material tochemically bond the two materials together through a covalent linkage.

[0068] Yet another preferred method of the present invention may beemployed to immobilize a least one biomolecule comprising at least oneisocyanate moiety on a biomaterial surface comprising at least oneunsubstituted amide moiety. The method includes the steps of: providinga biomaterial surface comprising an unsubstituted amide moiety,converting the amide moiety into an amine moiety using an amine formingagent to form an amine-functional material; combining theisocyanate-functional biomolecule with the amine-functional material tochemically bond the two materials together through a covalent linkage.

[0069] Another preferred method of the present invention may be employedto immobilize at least one biomolecule comprising at least onenegatively charged moiety, such as a phosphate moiety, a sulphate moietyor a carboxylate moiety, on a biomaterial surface comprising at leastone unsubstituted amide moiety. The method includes the steps of:providing a biomaterial surface comprising an unsubstituted amidemoiety, converting the amide moiety into an amine moiety using an amineforming agent to form an amine-functional material; combining thenegatively charged biomolecule with the amine-functional material tochemically bond the two materials together through an ionic linkage.

[0070] One preferred method of the present invention may be employed toimmobilize at least one biomolecule comprising at least one chemicalmoiety which is capable of forming a chemical bond with aguanidino-functional material (such as, for example, a 1,2-dicarbonylmoiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety)on a biomaterial surface comprising at least one unsubstituted amidemoiety. The method includes the steps of: providing a biomaterialsurface comprising an unsubstituted amide moiety, converting the amidemoiety into an amine moiety using an amine forming agent to form anamine-functional material; converting the amine-functional material intoa guanidino-functional material using a guanidino forming agent;combining the guanidino-functional material with a biomolecule capableof forming a chemical bond with a guanidino-functional material tochemically bond the two materials together through a chemical linkage.

[0071] Still another preferred method of the present invention may beemployed to immobilize a least one biomolecule comprising a least oneunsubstituted amide moiety on a biomaterial surface comprising a leastone chemical moiety which is capable of forming a chemical bond with anaminefunctional material (such as, for example, an aldehyde moiety, anepoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphatemoiety or a carboxylate moiety). The method includes the steps of:providing a biomolecule comprising an unsubstituted amide moiety,converting the amide moiety into an amine moiety using an amine formingagent to form an aminefunctional material; combining theamine-functional material with a biomaterial surface capable of forminga chemical bond with an aminefunctional material to chemically bond thetwo materials together through a chemical linkage.

[0072] Yet another preferred method of the present invention may beemployed to immobilize a least one biomolecule comprising a least oneunsubstituted amide moiety on a biomaterial surface comprising a leastone chemical moiety which is capable of forming a chemical bond with aguanidino-functional material (such as, for example, a 1,2-dicarbonylmoiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety).The method includes the steps of: providing a biomolecule comprising anunsubstituted amide moiety, converting the amide moiety into an aminemoiety using an amine forming agent to form an amine-functionalmaterial; converting the amine-functional material into aguanidino-functional material using a guanidino forming agent; combiningthe guanidino-functional material with a biomaterial capable of forminga chemical bond with a guanidino-functional material to chemically bondthe two materials together through a chemical linkage.

[0073] One preferred method of the present invention may be employed tocrosslink biomolecules, located in solution or on biomaterial surfaces,comprising at least one unsubstituted amide moiety and at least onechemical moiety which is capable of forming a chemical bond with anamine-functional material (such as, for example, an aldehyde moiety, anepoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphatemoiety or a carboxylate moiety). This method comprises the steps of:providing a biomolecule comprising an unsubstituted amide moiety,converting the amide moiety into an amine moiety using an amine formingagent to form an amine-functional biomolecule; allowing theamine-functional biomolecule to combine with a biomolecule capable offorming a chemical bond with an amine-functional biomolecule tochemically bond the two biomolecules together through a chemicallinkage, thereby forming a crosslinked material. This crosslinkedmaterial may be employed as a biomaterial or as a biomaterial coating.In addition, such crosslinked material may be further modified tocontain additional biomolecules. For example, biomolecules comprisingaldehyde moieties may be attached to residual amine moieties present inor on the surface of the crosslinked material.

[0074] Another preferred method of the present invention may be employedto crosslink biomolecules, located in solution or on biomaterialsurfaces, comprising at least one unsubstituted amide moiety and atleast one chemical moiety which is capable of forming a chemical bondwith a guanidino-functional material (such as, for example, a1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or acarboxylate moiety). This method comprises the steps of: providing abiomolecule comprising an unsubstituted amide moiety, converting theamide moiety into an amine moiety using an amine forming agent to forman amine-functional biomolecule; converting the amine-functionalbiomolecule into a guanidino-functional biomolecule using a guanidinoforming agent; allowing the guanidino-functional biomolecule to combinewith a biomolecule capable of forming a chemical bond with anguanidino-functional biomolecule to chemically bond the two biomoleculestogether through a chemical linkage, thereby forming a crosslinkedmaterial. This crosslinked material may be employed as a biomaterial oras a biomaterial coating. Additionally, such crosslinked material may befurther modified to contain additional biomolecules. For example,biomolecules comprising 1,2-dicarbonyl moieties may be attached toresidual guanidino moieties present in or on the surface of thecrosslinked material.

[0075] One example of a biomolecule of the present invention iscollagen. Collagen, which is found in connective tissue, has specialamino acids, one of which is 5-hydroxylysine which may be oxidized witha source of periodate, which may be provided as periodic acid or saltsthereof, such as sodium periodate, potassium periodate, or other alkalimetal periodates, to form a pendant aldehyde moiety. The resultantaldehyde moieties may be used to crosslink the collagen through bondsformed between the aldehydes and amines, for example, lysine amino acidresidues or modified asparagine amino acid residues, contained onneighboring collagen molecules. The resultant imine bonds may then bereduced using a mild reducing agent like sodium borohydride, sodiumcyanoborohydride, or amine boranes. These crosslinks may endow thecollagen biomaterial or biomaterial coating with desirable biologicaland/or physical properties such as mechanical strength,anti-immunogenicity, biostability, among others, without the use of acoupling agent. Thus, the method of the present invention eliminates theneed for using glutaraldehyde, a commonly used cytotoxic coupling agent,to crosslink the collagen to control its physical and biologicalproperties.

[0076] The aldehyde moieties formed by oxidation of collagen may also beused to couple a variety of amine-containing biomolecules to thecrosslinked collagen biomaterial or biomaterial coating. Also, theability to create aldehyde moieties along collagen molecules enablesthem to be covalently attached to amine containing biomaterial surfaces.Such collagen-coated biomaterial surfaces may be used, for example, ascell seeding surfaces, cell binding surfaces, cell separating surfaces,tissue fixation, collagen-coated stents, collagen-coated vascular graftsor collagen glues.

[0077] Other biomolecules, such as structural proteins, may becrosslinked to form a material that may be used as a biomaterial or abiomaterial coating. Also, additional biomolecules, as described herein,may be attached to residual amine moieties contained in or on afabricated crosslinked biomaterial or biomaterial coating, as describedherein. Alternatively, amine containing biomolecules may be attached toresidual aldehyde moieties contained in or on a fabricated crosslinkedbiomolecule biomaterial or biomaterial coating, as described herein.

[0078] An example of a glycoprotein that can be used in a number ofaspects of the present invention is fibrin(ogen). Fibrin(ogen), which isa structural protein, has oligosaccharides which can be oxidized with asource of periodate, which can be provided as periodic acid or saltsthereof, such as sodium periodate, potassium periodate, or other alkalimetal periodates, to form a pendant aldehyde moiety. The resultantaldehyde moieties can be used to crosslink the fibrin(ogen) throughbonds formed between the aldehydes and amines contained on neighboringfibrin(ogen) molecules. The resultant imine bonds can then be reducedusing a mild reducing agent like sodium borohydride, sodiumcyanoborohydride, or amine boranes. These crosslinks can endow thefibrinogen and/or fibrin (thrombin polymerized fibrinogen) biomaterialor biomaterial coating with desirable biological and/or physicalproperties such as mechanical strength, anti-immunogenicity,biostability, among others, without the use of a coupling agent. Thus,the method of the present invention eliminates the need for usingglutaraldehyde, a commonly used cytotoxic coupling agent, to crosslinkthe fibrinogen and/or fibrin to control its physical and biologicalproperties.

[0079] The aldehyde moieties formed by oxidation of fibrin(ogen) canalso be used to couple a variety of amine-containing biomolecules to thecrosslinked fibrin(ogen) biomaterial or biomaterial coating. Also, theability to create aldehyde moieties along fibrin(ogen) molecules enablesthem to be covalently attached to amine containing biomaterial surfaces.Such fibrinogen/fibrin coated biomaterial surfaces can be used, forexample, as cell seeding surfaces, cell binding surfaces, cellseparating surfaces, fibrinogen/fibrin-coated stents,fibrinogen/fibrin-coated vascular grafts or fibrinogen/fibrin glues.

[0080] Although the examples described below relate generally totreatment of polymeric films or tissue culture plates as substratesurfaces, those examples are merely illustrative and are intended tolimit in no way the scope of the present invention.

EXAMPLE 1 Periodate Oxidation of a Peptide Containing an N-terminalSerine Amino Acid Residue

[0081] Two biomolecules, a tripeptide made of three serine amino acidresidues and a dipeptide made of two lysine amino acid residues, bothobtained from Sigma Chemical Co. (St. Louis, Mo.), were incubated insodium metaperiodate (NaIO₄) also obtained from Sigma Chemical Co. (St.Louis, Mo.). The tripeptide, 0.90 mmoles, was incubated in the darkwhile shaking at room temperature for 3 hours in 10 ml deionized watercontaining 1.2 mmoles NaIO₄. The resultant solution, 2.5 ml, was addedto 2 ml of a solution containing 0.8 g NaOH, 0.2 g4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, which is available underthe trade designation PURPALD from Sigma Chemical Co. (St. Louis, Mo.),in 20 ml deionized water, and shaken vigorously for 15 minutes at roomtemperature. The dipeptide, 0.72 mmoles, was incubated in the dark whileshaking at room temperature for 3 hours in 10 ml deionized watercontaining 1.2 mmoles NaIO₄. The resultant solution, 10 ml (note thatthis amount is four times the amount used for the tripeptide), was thenadded to 2 ml PURPALD solution and shaken vigorously for 15 minutes atroom temperature. The resultant solutions were then analyzedspectrophotometrically at 550 nm. Dickinson and Jacobsen, Chem. Commun.,1719 (1970), described the specific and sensitive reaction of aldehydeswith PURPALD to yield purple-to-magenta-colored6-mercapto-5-triazolo-(4,3-b)-s-tetrazines which can be measuredspectrophotometrically at 550 nm. Sample absorbances obtained at 550 nmwere 0.04 for the dipeptide and 1.81 for the tripeptide, which indicatesthat only the tripeptide which contained an N-terminal serine wassuccessfully oxidized using periodate. The dipeptide of the two lysineamino acids lacked a 2-aminoalcohol moiety, that is a carbon-carbon bondbearing an amine moiety adjacent to a hydroxyl moiety.

EXAMPLE 2 Periodate Oxidation of a Peptide Containing an N-terminalThreonine Amino Acid Residue

[0082] A biomolecule, a dipeptide made of N-terminal threonine andleucine amino acid residues obtained from Sigma Chemical Co. (St. Louis,Mo.), was incubated in sodium metaperiodate (NaIO₄) also obtained fromSigma Chemical Co. (St. Louis, Mo.). The dipeptide, 4.3 mmoles, wasincubated in the dark while shaking at room temperature for 3 hours in10 ml deionized water containing 1.2 mmoles NaIO₄. The resultantsolution, 10 ml, was added to 2 ml of the PURPALD solution described inExample 1 and shaken vigorously for 15 minutes at room temperature.After the 15 minutes of shaking at room temperature, the resultantsolution was analyzed spectrophotometrically at 550 nm. Sampleabsorbance obtained at 550 nm was 0.62 indicating the periodate hadsuccessfully oxidized the N-terminal threonine amino acid present in thedipeptide, thereby forming an aidehyde moiety.

EXAMPLE 3 Periodate Oxidation of a Peptide Containing an N-terminalSerine Amino Acid Residue

[0083] A biomolecule, a pentapeptide made of N-terminal serine, asparticacid, glycine, arginine, and glycine amino acid residues obtained fromSigma Chemical Co. (St. Louis, Mo.), was incubated in sodiummetaperiodate (NaIO₄) also obtained from Sigma Chemical Co. (St. Louis,Mo.). The pentapeptide, 0.01 mmoles, was incubated in the dark whileshaking at room temperature for 3 hours in 2 ml deionized watercontaining 0.23 mmoles NaIO₄. The resultant solution, 10 ml, was addedto 2 ml of the PURPALD solution described in Example 1 and shakenvigorously for 15 minutes at room temperature. After the 15 minutes ofshaking at room temperature, the resultant solution was analyzedspectrophotometrically at 550 nm. Sample absorbance obtained at 550 nmwas 0.74, indicating the periodate had successfully oxidized theN-terminal serine amino acid residue present in the pentapeptide,thereby forming an aldehyde moiety.

EXAMPLE 4 Oxidation of Collagen

[0084] The biomolecule, mouse collagen, type IV, obtained from SigmaChemical Co. (St. Louis, MO), was oxidized with sodium metaperiodate(NaIO₄). Collagen type IV is known to mediate the attachment ofepithelial, endothelial, myoblasts and nerve cells in vivo and in vitro.Two collagen solutions were prepared by i) mixing half a vial ofcollagen with 56 mg NaO₄ in 5 ml deionized water and ii) mixing half avial of collagen in 5 ml deionized water. Both solutions were incubatedin the dark for 2 hours while shaking at room temperature. The resultantsolutions, 100 ml of each, were added to 2 ml the PURPALD solutiondescribed in Example 1 and shaken vigorously for 30 minutes at roomtemperature. After the 30 minutes of shaking at room temperature, theresultant solutions were analyzed spectrophotometrically at 550 nm. ThePURPALD solution was used as the blank. Sample absorbances obtained at550 nm were 0.03 for nonoxidized collagen and 0.25 for oxidizedcollagen, indicating the periodate had successfully oxidized thecollagen, thereby forming aldehyde moieties.

EXAMPLE 5 Attachment of Periodate Oxidized Biomolecules to AminatedSubstrates

[0085] One method for creating amines on substrate surfaces entailsgrafting substrate surfaces with acrylamide (AAm) andN-(3-aminopropyl)methacrylamide (APMA) monomers using ceric (CetV) ions.The Ce^(IV) ions create free radicals on ozone treated silicone andpolystyrene surfaces and untreated polyurethane surfaces which initiatethe graft copolymerization of the acrylamides. The amount of surfaceamination (the graft copolymerization of APMA and AAm) that takes placeon the substrate surface may be measured via staining with ponceau Sdye, a negatively charged dye molecule. This dye ionically associateswith the primary amines on the aminated surface. Following grafting, aperiodate oxidized biomolecule may be coupled to the amine containingderivatized substrate surface. A 2-aminoalcohol-containing biomoleculeis first oxidized with sodium metaperiodate (NaIO₄) forming a reactivealdehyde moiety. The aldehyde moiety is then used to covalently attachedthe biomolecule to the primary amine moiety present on the substratesurface. Sodium cyanoborohydride (NaCNBH₃) is then used to stabilize theimine linkages. Specific procedures required for each of these steps aredescribed below.

[0086] Polystyrene 24 well tissue culture plates were ozone treated byplacing the culture plates in an ozone reaction vessel for 30 minuteswhile oxygen, which contained ozone, was flowing at a rate of 1.3cm³/min. The oxygen containing ozone was created by flowing the oxygenthrough a corona discharge apparatus, which exposed the flowing oxygento an 8000V electrical potential. Following ozone treatment, the plateswere soaked in nitrogen purged deionized water for 30 minutes at roomtemperature. Following the 30 minute soak in nitrogen purged deionizedwater, the plates were grafted with acrylamide (Mm) andN-(3-aminopropyl)methacrylamide (APMA) monomers using Ce^(IV) ion. Thegrafting solution consisted of 40 g Mm, 10 g APMA, 50 g deionized watersolution, and 20 g Ce^(IV) ion solution. The Ce^(IV) ion solutionconsisted of 2.74 g ceric ammonium nitrate and 3.15 g nitric acid in 50ml deionized water. The plates were allowed to graft for 3 hours in a 65degrees Celsius nitrogen purged oven. Following grafting, the plates arerinsed vigorously with deionized water. The grafted plates were thentested with ponceau S dye. Following staining, the ponceau S dye wasreleased from the surface using a 1% sodium dodecyl sulphate (SDS)solution and quantified spectrophotometrically at 520 nm. Sampleabsorbances obtained at 520 nm were 0.00 for nonderivatized plates and1.44 for surface-derivatized plates. As the results demonstrate, thesurfacederivatized plates contain primary amines on their surfaces.

[0087] The 2-aminoalcohol moiety of a peptide may be oxidized using theprocedure of Example 1. Sodium cyanoborohydride (1 mg/ml) then is addedto the oxidized peptide solution. The resultant solution then isimmediately added to each of the amine containing surface-derivatizedtissue culture plate wells (approximately 1 ml solution/well). Theoxidized peptide is then incubated in the derivatized tissue cultureplate wells overnight at room temperature. Following incubation, thewells are vigorously rinsed with phosphate buffered saline (PBS)solution.

[0088] Polyurethane film samples were cut into 1.4 cm diameter disks.Sample disks were grafted with AAm and APMA monomers using Ce^(IV) ion.The sample disks were allowed to graft 1 hour at room temperature.Following grafting, the sample disks were rinsed vigorously withdeionized water. Again, the 2-aminoalcohol moiety of a peptide can beoxidized as previously described. Sample disks are then exposed to theoxidized peptide solution. Sodium cyanoborohydride is then added (1mg/ml) and the resultant solution and sample disks are incubatedovernight at room temperature. Following incubation, the polyurethanesample disks are vigorously rinsed with PBS.

EXAMPLE 6 Crosslinking of Collagen

[0089] A biomolecule such as collagen, type IV, may be oxidized withsodium metaperiodate (NaIO₄). A collagen solution may be prepared bymixing half a vial of collagen with 56 mg NaIO₄ in 5 ml deionized. Thesolution may be incubated in the dark for 2 hours while shaking at roomtemperature. The oxidized collagen molecules are then allowed to formcrosslinks, thereby bonding the molecules together through iminemoieties. An imine moiety is formed from an aldehyde moiety of onecollagen molecule reacting with an amine moiety of a neighboringcollagen molecule. The imine linkages are then stabilized by reactingthe imine moieties with sodium cyanoborohydride (1 mg/ml) to formsecondary amine linkages. The resultant crosslinked material may beemployed as a biomaterial or as a biomaterial coating.

EXAMPLE 7 Periodate Oxidation of Bovine Fibrinogen

[0090] The glycoprotein bovine fibrinogen obtained from Sigma ChemicalCo. (St. Louis, Mo.) was incubated in sodium metaperiodate (NaIO₄) alsoobtained from Sigma Chemical Co. (St. Louis, Mo.). The following fourfibrinogen solutions were prepared to investigate the oxidation offibrinogen with varying amounts of periodate: (1) 0.03 mM fibrinogen,0.2 mM NaIO₄, 0.008 M Na₂HPO₄, 0.002 M KH₂PO₄, 0.14 M NaCl, pH 7.4; (2)0.03 mM fibrinogen, 0.1 mM NaIO₄, 0.008 M Na₂HPO₄, 0.002 M KH₂PO₄, 0.14M NaCl, pH 7.4; (3) 0.03 mM fibrinogen, 0.05 mM NaIO₄, 0.008 M Na₂HPO₄,0.002 M KH₂PO₄, 0.14 M NaCl, pH 7.4; and (4) 0.03 mM fibrinogen, 0.008 MNa₂HPO₄, 0.002 M KH₂PO₄, 0.14 M NaCl, pH 7.4. The four fibrinogensolutions were incubated in the dark for 2 hours while shaking at roomtemperature. The resultant solutions, 500 μl of each, were added to 2 mlof a solution containing 0.8 g NaOH, 0.2 g4-amino-3-hydrazino-5-mercato-1,2,4-triazole, which is available underthe trade designation PURPALD from Sigma Chemical Co. (St. Louis, Mo.),in 20 ml deionized water, and shaken vigorously for 15 minutes at roomtemperature. Dickinson and Jacobsen, Cem. Commun., 1719 (1970),described the specific and sensitive reaction of aldehydes with PURPALDto yield purple-to-magenta-colored6-mercapto-s-triazolo-(4,3-b)-s-tetrazines. After the 15 minutes ofshaking at room temperature, the resultant solutions were analyzedspectrophotometrically at 550 nm. Sample 4 was used as the blank. Sampleabsorbances obtained at 550 nm were 0.54 for sample 1, 0.53 for sample 2and 0.51 for sample 3, which indicates that for all samples thefibrinogen was successfully oxidized forming aldehyde groups.

EXAMPLE 8 Periodate Oxidation of Bovine Vitronectin

[0091] The glycoprotein bovine vitronectin obtained from Sigma ChemicalCo. (St. Louis, Mo.) was incubated in sodium metaperiodate (NaIO₄) alsoobtained from Sigma Chemical Co. (St. Louis, Mo.). The following twovitronectin solutions were prepared: (1) 0.001 mM vitronectin, 0.05 MNaIO₄ and (2) 0.001 mM vitronectin. Both solutions were incubated in thedark for 2 hours while shaking at room temperature. The resultantsolutions, 100 μl of each, were added to 2 ml PURPALD solution describedin Example 1, and shaken vigorously for 30 minutes at room temperature.After the 30 minutes of shaking at room temperature, the resultantsolutions were analyzed spectrophotometrically at 550 nm. The PURPALDsolution was used as the blank. Sample absorbances obtained at 550 nmwere 0.09 for sample 1 and 0.04 for sample 2, which indicated thatvitronectin was successfully oxidized with forming aldehyde groups.

EXAMPLE 9 Periodate Oxidation of Bovine Fibronectin

[0092] The glycoprotein bovine fibronectin obtained from Sigma ChemicalCo. (St. Louis, Mo.) was incubated in sodium metaperiodate (NaIO₄) alsoobtained from Sigma Chemical Co. (St. Louis, Mo.). The following twofibronectin solutions were prepared: (1) 0.002 mM fibronectin, 0.05 MNaIO₄, 0.5 M NaCl, 0.05 M Tris, pH 7.5; and (2) 0.002 mM fibronectin,0.5 M NaCl, 0.05 M Tris, pH 7.5. Both solutions were incubated in thedark for 2 hours while shaking at room temperature. The resultantsolutions, 100 μl of each, were added to 2 ml PURPALD solution describein Example 1 and shaken vigorously for 30 minutes at room temperature.After 30 minutes of shaking at room temperature, the resultant solutionswere analyzed spectrophotometrically at 550 nm. Following an initialanalysis, sample 1 was observed to contain to many aldehydes to measure.Therefore, sample 1 was diluted 1:50 in deionized water to achieve ameasurable amount of aldehydes contained in the sample solution. ThePURPALD solution was used as the blank. Sample absorbances obtained at550 nm were 0.81 for sample 1 and 0.0 for sample 2, which indicate thatthe fibronectin in sample 1 was successfully oxidized forming aldehydegroups. Fibronectin in sample 2 was not oxidized due to the omission ofperiodate.

EXAMPLE 10 Attachment of Fibronectin to Aminated Substrates

[0093] Fibronectin was covalently attached to a substrate surface. Theattachment technique began with the graft copolymerization of acrylamide(AAm) and N-(3-aminopropyl)methacrylamide (APMA) monomers onto an ozonetreated polystyrene tissue culture plate with ceric (Ce^(IV)) ions. TheCe^(IV) ions create free radicals on the ozone treated surface whichinitiate the graft copolymerization of the acrylamides. The amount ofsurface amination (the graft copolymerization of APMA and AAm) that tookplace on the substrate surface was measured via staining with ponceau Sdye, a negatively charged dye molecule. Following grafting, fibronectinwas coupled to the amine containing derivatized substrate surface.Fibronectin was first oxidized with sodium metaperiodate (NaIO₄) formingreactive aldehyde groups. These aldehyde groups were then used tocovalently attached fibronectin to the primary amino groups present onthe substrate surface. Sodium cyanoborohydride (NaCNBH₃) was then usedto stabilize the imine linkages. The specific procedures for each ofthese steps are described below.

[0094] Polystyrene 24 well tissue culture plates were ozone treated byplacing the culture plates in an ozone reaction vessel for 30 minuteswhile oxygen, which contained ozone, was flowing at a rate of 1.3cm³/min. The oxygen containing ozone was created by flowing the oxygenthrough a corona discharge apparatus, which exposes the flowing oxygento an 8000V electrical potential. Following ozone treatment, the plateswere soaked in nitrogen purged deionized water for 30 minutes at roomtemperature. Following the 30 minute soak in nitrogen purged deionizedwater, the plates were grafted with acrylamide (AAm) andN-(3-aminopropyl)methacrylamide (APMA) monomers (Eastman Kodak Co.,Rochester, N.Y.) using ammonium cerium (IV) nitrate (Aldrich ChemicalCo., Milwaukee, Wis.). The grafting solution consisted of 11.2 M AAm,1.1 M APMA, 400 mM nitric acid and 40 mM ammonium cerium (IV) nitrate indeionized water. The plates were allowed to graft for 3 hours in a 65°C. nitrogen purged oven. Following grafting the plates are rinsedvigorously with deionized water. The grafted plates were then testedwith ponceau S dye. Following staining, the ponceau S dye was releasedfrom the surface using a 1% sodium dodecyl sulphate (SDS) solution andquantified spectrophotometrically at 520 nm. Sample absorbances obtainedat 520 nm were 0.00 for nonderivatized plates and 1.44 forsurface-derivatized plates. As the results demonstrate, thesurface-derivatized plates contain primary amines on their surfaces.

[0095] Bovine fibronectin obtained from Sigma Chemical Co. (St. Louis,Mo.) was then incubated in sodium metaperiodate (NaIO₄) also obtainedfrom Sigma Chemical Co. (St. Louis, Mo.). The following fibronectinsolution was prepared: 0.002 mM fibronectin, 0.05 M NaIO₄, 0.5 M NaCl,0.05 M Tris, pH 7.5. The solution was incubated in the dark for 2 hourswhile shaking at room temperature. Sodium cyanoborohydride (1 mg/ml) wasthen added to the fibronectin solution. The resultant solution wasimmediately added to each of the amine containing surface-derivatizedtissue culture plate wells (approximately 1 ml solution/well). Thefibronectin solution incubated in the derivatized tissue culture platewells overnight at room temperature. Following incubation, the wellswere then vigorously rinsed with phosphate buffered saline (PBS)solution. The attachment of fibronectin to the amine containingsurface-derivatized tissue culture plate surfaces was assessed usingtoluidine blue dye, a positively charged dye molecule. This dyeionically associates with the negative charges on a substrate surface.Therefore, the binding of toluidine blue dye to thefibronectin-derivatized surface is due to fibronectin's negativecharges. The wells of each plate were filled with a 1% toluidine bluedye in deionized water solution. After a 5 minute incubation at roomtemperature, the dye solution was removed and the wells were thoroughlyrinsed with PBS. The surface associated dye in each well was then elutedby mechanically shaking the plates in a 1% SDS in deionized watersolution overnight. The amount of dye eluted from the wells was thendetermined spectrophotometrically at 630 nm. Sample absorbances obtainedat 630 nm were 0.05 for the nonderivatized sample plate, 0.54 for theAAm/APMA-derivatized sample plate and 1.83 for thefibronectin-derivatized sample plate, which indicate that thefibronectin was successfully oxidized and then covalently attached tothe substrate surface.

EXAMPLE 11 ELISA and Cellular Adherence to Fibronectin Coupled Surfaces

[0096] Polyurethane in the form of Pellethane 2363-55D was obtained fromDow Chemical Co. (Midland, Mich.) and extruded into film. The film wasthen cut into 1 cm sample disks. Sample disks were then cleansed withethanol and surface grafted with AAm and APMA monomers using Ce^(IV)ion. The grafting solution consisted of 11.2 M AAm, 1.1 M APMA, 400 mMnitric acid and 40 mM ammonium cerium (IV) nitrate in deionized water.The sample disks were placed into the grafting solution and allowed tograft for 1 hour at room temperature. Following grafting, the sampledisks were thoroughly washed with deionized water. Fibronectin was thencoupled to the resultant APMA/Mm surface-derivatized sample disks viatwo methods.

[0097] The first method or peroxide method included the oxidation offibronectin by sodium metaperiodate. Fibronectin (0.1 mg/ml) was exposedin the dark to a 1 μg/ml sodium metaperiodate in deionized watersolution for 3 hours at room temperature. The APMA/AAm-derivatizedsample disks were then placed into the oxidized fibronectin solution for24 hours at room temperature. Sample disks were then thoroughly rinsedwith deionized water. The samples were then incubated for 24 hours atroom temperature in a 3 mg/ml sodium cyanoborohydride in deionized watersolution. Sample disks were then thoroughly rinsed with deionized water.

[0098] The second method used glutaraldehyde as a coupling agent. Themethod included soaking the APMA/AAm-derivatized sample disks in a 2%glutaraldehyde in deionized water solution for 2 hours at roomtemperature. Sample disks were then thoroughly rinsed with deionizedwater. Following rinsing, the sample disks were then incubated in a 0.1mg/ml fibronectin in deionized water solution for 24 hours at roomtemperature. Sample disks were then thoroughly rinsed with deionizedwater. The sample disks were then incubated for 24 hours at roomtemperature in a 3 mg/ml sodium cyanoborohydride in deionized watersolution. Sample disks were then thoroughly rinsed with deionized water.

[0099] An enzyme linked immunosorbent assay (ELISA) was then performedto determine the ability of an antibody to recognize the fibronectinwhich had been coupled to the sample surfaces. Sample disks were washedfor 20 minutes at room temperature with wash buffer (pH 7.4) consistingof 10 mM Tris, 0.15 M NaCl and 0.05% Tween. Sample disks were thenincubated at 37° C. for 30 minutes in blocking buffer (pH 7.4)consisting of 10 mM Tris, 0.15 M NaCl, 0.05% Tween and 0.05% gelatinfollowed by three 10 minute washes with wash buffer. Next, sample diskswere incubated at 37° C. for 1 hour in a primary antibody solution (pH7.4) consisting of 10 mM Tris, 0.15 M NaCl and 2 μg/ml mouse monoclonalanti-fibronectin antibody (Sigma Chemical Co., St. Louis, Mo.). Sampledisks were then rinsed thrice (10 minutes per wash) with wash buffer.Next, sample disks were incubated at 37° C. for 1 hour in aperoxidase-labeled secondary antibody solution (pH 7.4) consisting of 10mM Tris, 0.15 M NaCl and 0.5 ng/ml anti-mouse IgG peroxidase antibodyconjugate (Sigma Chemical Co., St. Louis, Mo.). Sample disks were thenrinsed thrice (10 minutes per wash) with wash buffer. Sample disks werethen incubated for 15 minutes at room temperature in a phosphate-citratebuffer (pH 5.0) containing 0.4 mg/ml o-phenyidiamine dihydrochloride and0.2 μl/ml 30% hydrogen peroxide. The phosphate-citrate buffer consistedof 50 mM dibasic sodium phosphate and 25 mM citric acid in deionizedwater. Following the 15 minute incubation, the peroxide reaction wasstopped with 3 M HCl and the absorbance of the resultant solution wasmeasured spectrophotometrically at 492 nm. The APMA/AAm-derivatizedsample disks were used as controls for this experiment. Sampleabsorbances obtained from the spectrophotometric analysis were0.016±0.038 for APMA/Am-derivatized samples which containedglutaraldehyde coupled fibronectin and 0.204±0.068 forAPMA/AAm-derivatized samples which contained periodate oxidizedfibronectin. The results indicate that the periodate oxidation methodwas more successful at attaching fibronectin to the sample surfaces.

[0100] A cellular adherence assay was also performed to determine theability of cells to adhere to fibronectin-derivatized sample surfaces.Sample disks were incubated for 1 hour at 37° C. in a blocking bufferconsisting of 2 mg/ml ovalbumin in phosphate buffered saline (PBS), pH7.4. Mouse fibroblasts (C3T3) obtained from American Type CultureCollection (Rockville, Md.) and maintained in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal bovine serum wereharvested using trypsin:EDTA and resuspended in serum-free DMEMcontaining 2 mg/ml ovalbumin. The cells were then washed twice, countedand resuspended to a final density of 5×10⁴ cells/ml in serum-free DMEMcontaining 2 mg/ml ovalbumin. Sample disks were then incubated in thecell suspension for 1 hour at 37° C. Nonadherent cells were removed by aPBS wash. Sample disks were then fixed in 3% paraformaldehyde solutionfor 30 minutes. Adherent cells were then stained with a stainingsolution consisting of 1% toluidine blue dye and 3% paraformaldehyde inPBS. Following staining, sample surfaces were then examined for cellularadherence using a light microscope. Upon examination,APMA/AAm-derivatized samples and APMA/Am-derivatized samples whichcontained glutaraldehyde coupled fibronectin appeared to have noadherent cells. In contrast, cells appeared adherent toAPMA/AAm-derivatized samples which contained periodate oxidizedfibronectin.

EXAMPLE 12 Crosslinking of Fibrinogen

[0101] Porcine fibrinogen obtained from Sigma Chemical Co. (St. Louis,Mo.) was incubated in sodium metaperiodate (NaIO₄) also obtained fromSigma Chemical Co. (St. Louis, Mo.) and sodium cyanoborohydride(NaCNBH₃) obtained from Aldrich Chemical Co. (Milwaukee, Wis.). Thefollowing fibrinogen solution was prepared: 0.03 mM fibrinogen, 0.02 MNaIO₄, 0.02 M NaCNBH₃, 0.008 M Na₂HPO₄, 0.002 M KH₂PO₄, 0.14 M NaCl, pH7.4. The solution was then shaken vigorously and placed into a 24 welltissue culture plate (approximately 1 ml of fibrinogen solution/well).The plate was then incubated in the dark for 2 hours while shaking atroom temperature. After 2 hours, the solution was observed to havebecome cloudy and very viscous indicating the fibrinogen hadcrosslinked. The sample was then shaken for an additional 22 hours inthe dark. Following incubation, the crosslinked fibrinogen was testedfor residual aldehydes using the PURPALD solution describe in Example 1.The results of the PURPALD assay demonstrated few residual aldehydeswere present which indicated the formation of covalent crosslinksbetween the aldehydes and the amines present along the fibrinogenmolecules.

[0102] The following bovine fibrinogen (Sigma Chemical Co., St. Louis,Mo.) solution was prepared: 0.02 mM fibrinogen, 0.008 M Na₂HPO₄, 0.002 MKH₂PO₄, 0.14 M NaCl, pH 7.4. Following preparation, the solution wasdivided into four equal portions. Sodium metaperiodate (0.05 mM) wasthen added to samples 3 and 4. All four fibrinogen solutions were thenincubated in the dark for 2 hours while shaking at room temperature.Next, 0.02 mM NaCNBH₃ was added to samples 2 and 4. Again, all fourfibrinogen solutions were allowed to react for 2 hours while shaking atroom temperature. The samples, 50 μl of each, were then placed into 450μl of SDS-PAGE buffer solution consisting of 62.5 mM Tris-HCL, 5%b-mercaptoethanol, 10% glycerol and 2.3% SDS. Samples were then boiledfor 3 minutes. The samples, 10 μl of each, were then loaded onto a 4-15%gradient gel and SDS-PAGE was performed according to the proceduresdescribed in O'Farrell, “High Resolution Two-dimensional Electrophoresisof Proteins”, J. Biol. Chem. 250, 4007-4021 (1974). Followingelectrophoresis, the gel was stained with Coomassie Brilliant Blue, andthe identity of the eluted proteins was determined by reference tomolecular weight standards included on the gel. The results fromSDS-PAGE indicated that the fibrinogen molecules in sample 4 had formedstable covalent crosslinks. In contrast, the results demonstrated thatthe fibrinogen molecules in the samples which contained no NaIO₄ hadformed no crosslinks.

[0103] It will be appreciated by those skilled in the art that while theinvention has been described above in connection with particularembodiments and examples, the invention is not necessarily so limited,and that numerous other embodiments, examples, uses, modifications anddepartures from the embodiments, examples and uses are intended to beencompassed by the claims attached hereto. The entire disclosure of eachpatent and publication cited herein is incorporated by reference, as ifeach such patent or publication were individually incorporated byreference herein.

We claim:
 1. A method of forming a coating on a surface of a medicaldevice, the coating imparting improved biocompatibility characteristicsto the surface, the method comprising the steps of: (a) providing themedical device, the device having a biomaterial disposed on or forming asurface thereof, the biomaterial comprising an unsubstituted amidemoiety; (b) combining the amide moiety with an amine forming agent toform an amine-functional surface; (c) providing a biomolecule, thebiomolecule comprising a chemical moiety selected from the groupconsisting of an aldehyde moiety formed by combining a periodate with a2-aminoalcohol moiety, an aldehyde moiety formed by combining aperiodate with a 1,2-dihydroxy moiety, an epoxide moiety, an isocyanatemoiety, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moietyand a carboxylate moiety; and (d) combining the chemical moiety with thesurface to form a chemical bond, the chemical bond immobilizing thebiomolecule on the surface, the immobilized biomolecule forming thecoating.
 2. The method of claim 1 wherein the device is selected fromthe group consisting of a blood-contacting medical device, atissue-contacting medical device, a bodily fluid-contacting medicaldevice, an implantable medical device, an extracorporeal medical device,a blood oxygenator, a blood pump, tubing for carrying blood, anendoprosthesis medical device, a vascular graft, a stent, a pacemakerlead, a heart valve, temporary intravascular medical device, a catheterand a guide wire.
 3. The method of claim 1 wherein the biomaterialcomprises an amino acid residue.
 4. The method of claim 1 wherein theamine forming agent is selected from the group consisting of bromine,bromide, bromite, hypobromite, chlorine, chloride, chlorite,hypochlorite, lead tetraacetate, benzyltrimethylammonium tribromide,[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene andiodosylbenzene.
 5. The method of claim 1 wherein the biomolecule isselected from the group consisting of an anticoagulant agent, anantithrombotic agent, a clotting agent, a platelet agent, a blood agent,an anti-inflammatory, an antibody, an antigen, an immunoglobulin, adefense agent, an enzyme, a hormone, a growth factor, aneurotransmitter, a cytokine, a regulatory agent, a transport agent, afibrous agent, a protein, avidin, a glycoprotein, a globular protein, astructural protein, a membrane protein, a cell attachment protein, apeptide, a glycopeptide, a structural peptide, a membrane peptide, acell attachment peptide, a proteoglycan, a toxin, an antibiotic agent,antibacterial agent, antimicrobial agent, a polysaccharide, acarbohydrate, a fatty acid, a catalyst, a drug, biotin, a vitamin, a DNAsegment, a RNA segment, a nucleic acid, a lectin, a dye and a ligand. 6.The method of claim 1 wherein the biomolecule is a naturally occurringbiomolecule.
 7. The method of claim 1 wherein the biomolecule is achemically synthesized biomolecule.
 8. The method of claim 1 wherein thebiomolecule comprises an amino acid residue.
 9. The method of claim 1wherein the periodate comprises at least one of a periodic acid, asodium periodate, an alkali metal periodate, and a potassium periodate.10. The method of claim 1 comprising the further step of combining atleast one reducing agent selected from the group consisting of sodiumborohydride, sodium cyanoborohydride and amine borane.
 11. The method ofclaim 1 comprising the further step of combining the amine-functionalsurface with a guanidino forming agent to form a guanidino-functionalsurface.
 12. The method of claim 11 comprising the further step ofcombining a stabilizing agent.
 13. The method of claim 12 wherein thestabilizing agent is a borate ion.
 14. The method of claim 11 whereinthe guanidino forming agent is selected from the group consisting ofS-ethylthiouronium bromide, S-ethylthiouronium chloride,O-methylisourea, O-methylisouronium sulfate, O-methylisourea hydrogensulfate, S-methylisothiourea, 2-methyl-1-nitroisourea,aminoiminomethanesulfonic acid, cyanamide, cyanoguanide, dicyandiamide,3,5-dimethyl-1-guanylpyrazole nitrate and 3,5-dimethyl pyrazole.
 15. Themethod of claim 1 wherein at least a portion of the surface forms atleast one of a tube, a rod, a membrane, a balloon, a bag, a sheet, astring, a suture, a fiber and a mesh.
 16. The method of claim 1 whereinthe surface comprises at least one of a biocompatible material selectedfrom the group consisting of a metal, titanium, titanium alloy,tin-nickel alloy, a shape memory alloy, aluminum oxide, platinum,platinum alloy, stainless steel, MP35N stainless steel, elgiloy,stellite, pyrolytic carbon, silver carbon, glassy carbon, polyamide,polycarbonate, polyether, polyester, polyolefin, polyethylene,polypropylene, polystyrene, polyurethane, polyvinylchloride,polyvinylpyrrolidone, silicone elastomer, fluoropolymer, polyacrylate,polyisoprene, polytetrafluoroethylene, rubber, ceramic, hydroxapatite,human protein, human tissue, animal protein, animal tissue, bone, skin,tooth, collagen, laminin, elastin, fibrin, wood, cellulose, compressedcarbon and glass.
 17. A method of forming a coating on a surface of amedical device, the coating imparting improved biocompatibilitycharacteristics to the surface, the method comprising the steps of: (a)providing the medical device, the device having a suitable biomaterialdisposed on or forming a surface thereof, a chemical moiety beingdisposed on the surface, the chemical moiety being selected from thegroup consisting of an aldehyde moiety, an epoxide moiety, an isocyanatemoiety, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moietyand a carboxylate moiety; (b) providing a biomolecule, the biomoleculecomprising an unsubstituted amide moiety; (c) combining the amide moietywith an amine forming agent to form an amine-functional biomolecule; and(d) combining the biomolecule with the surface to form a chemical bond,the chemical bond immobilizing the biomolecule on the surface, theimmobilized biomolecule forming the coating.
 18. The method of claim 17wherein the device is selected from the group consisting of ablood-contacting medical device, a tissue-contacting medical device, abodily fluid-contacting medical device, an implantable medical device,an extracorporeal medical device, a blood oxygenator, a blood pump,tubing for carrying blood, an endoprosthesis medical device, a vasculargraft, a stent, a pacemaker lead, a heart valve, temporary intravascularmedical device, a catheter and a guide wire.
 19. The method of claim 17wherein the biomaterial comprises an amino acid residue.
 20. The methodof claim 17 wherein the aldehyde moiety is formed by combining aperiodate with a 2-aminoalcohol moiety.
 21. The method of claim 17wherein the aidehyde moiety is formed by combining a periodate with a1,2-dihydroxy moiety.
 22. The method of claim 17 wherein the periodatecomprises at least one of a periodic acid, a sodium periodate, an alkalimetal periodate, and a potassium periodate.
 23. The method of claim 17wherein the biomolecule is selected from the group consisting of ananticoagulant agent, an antithrombotic agent, a clotting agent, aplatelet agent, a blood agent, an anti-inflammatory, an antibody, anantigen, an immunoglobulin, a defense agent, an enzyme, a hormone, agrowth factor, a neurotransmitter, a cytokine, a regulatory agent, atransport agent, a fibrous agent, a protein, avidin, a glycoprotein, aglobular protein, a structural protein, a membrane protein, a cellattachment protein, a peptide, a glycopeptide, a structural peptide, amembrane peptide, a cell attachment peptide, a proteoglycan, a toxin, anantibiotic agent, antibacterial agent, antimicrobial agent, apolysaccharide, a carbohydrate, a fatty acid, a catalyst, a drug,biotin, a vitamin, a DNA segment, a RNA segment, a nucleic acid, alectin, a dye and a ligand.
 24. The method of claim 17 wherein thebiomolecule is a naturally occurring biomolecule.
 25. The method ofclaim 17 wherein the biomolecule is a chemically synthesizedbiomolecule.
 26. The method of claim 17 wherein the biomoleculecomprises an amino acid residue.
 27. The method of claim 17 wherein theamine forming agent is selected from the group consisting of bromine,bromide, bromite, hypobromite, chlorine, chloride, chlorite,hypochlorite, lead tetraacetate, benzyltrimethylammonium tribromide,[bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene andiodosylbenzene.
 28. The method of claim 17 comprising the further stepof combining at least one reducing agent selected from the groupconsisting of sodium borohydride, sodium cyanoborohydride and amineborane.
 29. The method of claim 17 comprising the further step ofcombining the amine-functional biomolecule with a guanidino formingagent to form a guanidino-functional biomolecule.
 30. The method ofclaim 29 comprising the further step of combining a stabilizing agent.31. The method of claim 30 wherein the stabilizing agent is a borateion.
 32. The method of claim 29 wherein the guanidino forming agent isselected from the group consisting of S-ethylthiouronium bromide,S-ethylthiouronium chloride, O-methylisourea, O-methylisouroniumsulfate, O-methylisourea hydrogen sulfate, S-methylisothiourea,2-methyl-1-nitroisourea, aminoiminomethanesulfonic acid, cyanamide,cyanoguanide, dicyandiamide, 3,5-dimethyl-1-guanylpyrazole nitrate and3,5-dimethyl pyrazole.
 33. The method of claim 17 wherein at least aportion of the surface forms at least one of a tube, a rod, a membrane,a balloon, a bag, a sheet, a string, a suture, a fiber and a mesh. 34.The method of claim 17 wherein the surface comprises at least one of abiocompatible material selected from the group consisting of a metal,titanium, titanium alloy, tin-nickel alloy, a shape memory alloy,aluminum oxide, platinum, platinum alloy, stainless steel, MP35Nstainless steel, eigiloy, stellite, pyrolytic carbon, silver carbon,glassy carbon, polyamide, polycarbonate, polyether, polyester,polyolefin, polyethylene, polypropylene, polystyrene, polyurethane,polyvinylchloride, polyvinylpyrrolidone, silicone elastomer,fluoropolymer, polyacrylate, polyisoprene, polytetrafluoroethylene,rubber, ceramic, hydroxapatite, human protein, human tissue, animalprotein, animal tissue, bone, skin, a tooth, collagen, laminin, elastin,fibrin, wood, cellulose, compressed carbon and glass.